The first step in the isolation of platelets for analysis is generally the separation of platelets from whole blood through centrifugation. This results in a platelet rich plasma. The abundance of plasma proteins is extremely high compared to the platelet proteome so the next phase of preparing platelets is to attempt to separate them from plasma proteins. One method commonly used to prepare a plasma-free platelet suspension involves a further centrifugation step. Under higher forces than in the initial centrifugation of the whole blood, the platelets can be pelleted and then re-suspended in a suitable buffer. The mechanical stress induced during centrifugation means that the platelets can become easily activated which results in exocytosis of secretion vesicles and granules together with a number of other biochemical changes and a change in shape. If fresh platelet samples are frozen for storage, subsequent thawing or freeze-thaw cycles can cause lysis of the platelet membranes releasing contents into the surrounding medium.
An alternative method commonly used to avoid the mechanical stress imposed by centrifugation based methods of separation is gel filtration. This consists of passing platelet-rich plasma (PRP) through a column of, for example, Sepharose 2B or Biogel A-150 which can separate platelet proteins from plasma proteins according to their size (the platelets pass through the material due to their greater mass and the plasma proteins are generally retained in the micro-pores). Compared to the centrifuged based methods separation is rapid with minimal loss of platelets from the initial PRP. However gel filtration may not eliminate all larger molecular weight plasma proteins and can be more susceptible to methodological errors (Cazenave et al, 2004).
Platelet activation/lysis is a constant problem in the preparation of platelet samples for protein analysis, as due to their highly sensitive nature they are susceptible to a number of stimuli throughout the process, from blood collection to subsequent preparation. Activation/lysis often leads to exocytosis of proteins of diagnostic interest thus potentially decreasing the value of platelets obtained. Therefore it is important to take all necessary precautions to prevent preparation induced activation/lysis while at the same time maintaining platelet yield for proteomic studies. A further problem is often encountered when an immunoassay is employed to detect platelet proteins after processing, detergents (for example, sodium dodecyl sulphate (SDS)) which are used to lyse the platelets can also disrupt epitopes as well as denature antibodies used in detection leading to distorted results (Geumann et al, 2010). Development of a platelet preparation method which addresses these problems would be extremely beneficial in the field of platelet protein immunoassays.